Project Summary Abstract. Human endogenous retrovirus type K (HERV-K) is an ancient retrovirus that has become embedded in the human genome, but it has been silenced as a result of accumulation of mutations throughout evolution. Our previous studies have established that HERV-K is overexpressed in breast cancer and other cancers, but is not expressed in normal tissues, making it a particularly unique therapeutic target. We also recently reported that HERV-K regulates expression of the Ras oncoprotein in breast and other cancers. Although activated Ras mutant proteins are the most prevalent oncogenes in human cancers, Ras is considered to be ?undruggable,? and has not been effectively targeted in the clinic. However, the mechanism by which endogenous retroviruses impact expression and/or activation of Ras is unknown, and this project is designed to yield insights into the biological significance of this interaction. The central hypothesis tested in our proposal is that HERV-K interacts with components of Ras signaling pathways to alter expression of Ras in breast cancer cells, primarily triple negative breast cancer (TNBC). At the mechanistic level in Aim 1 we will: 1. Determine which Ras signaling proteins interact with HERV-K in TNBC cells, with and without a. HERV-K knockdown and overexpression b. inhibitors of three key breast cancer signaling pathways: Ras/ERK, TGF?, and PI3K/AKT 2. Evaluate a feed-forward mechanism that accelerates Ras activation in breast cancer cells. The second Aim will evaluate the effectiveness of HERV-K specific chimeric antigen receptor (K-CAR) T cells in killing HERV-K positive TNBC cells. Our previous studies showed that K-CAR T cells prepared from breast cancer patients were cytotoxic toward breast cancer cells and inhibited cell growth, but normal breast cells were unaffected. Tumor growth and tumor weight was also decreased in xenograft models bearing human breast cancer cells, and tumor metastasis was decreased in this model. Of interest, expression of HERV-K in these breast tumors correlated with expression of the oncoprotein Ras. K-CAR T cells could thus have a dual role in breast cancer: lyse the tumor cells and block Ras expression. We propose to evaluate both of these roles in an effort to maximize K-CAR efficacy in treating TNBC patients. We will evaluate cytotoxicity of K-CAR T cells prepared from TNBC patients toward breast cancer cells, and will use a TNBC patient- derived xenograft (PDX) model to determine efficacy of our K-CAR in tumor growth prevention in vivo. In addition, we will evaluate combined therapies of K-CAR plus Ras/ERK, TGF?, or PI3K/AK pathway inhibitors. The objective of Aim 2 is to target two major breast cancer pathways, one with K-CAR which simultaneously lyses tumors and targets Ras signaling, and a second with small molecule inhibitors to target an additional pathway. We expect to show an additive or perhaps even a synergistic effect from the combined therapy, with a goal of providing a new immunotherapeutic opportunity for TNBC patients.